The removal of introns by RNA splicing is an essential step in the expression of almost all human genes. Many nascent transcripts are subject to alternative splicing, which provides a means for making more than one protein from a single gene. Such alternative splicing can be either developmentally or tissue-specifically controlled. Whether or not an RNA is spliced is also subject to regulation: controlling the level of spliced and unspliced viral RNA in the cytoplasm is critical to the replication of HIV. Thus a detailed knowledge of the splicing process will be essential for better understanding of the basic mechanisms of gene expression, as well as organismal development, oncogenesis and the progression of retroviral infection. Because spliceosomal processing and the self-catalyzed excision of group II introns occur via the same two-step pathway, the two intron types are thought to be evolutionarily related. Therefore, the spliceosome has long been suspected to be an RNA catalyst. However, the precise makeup of the catalytic center(s) has remained elusive, primarily because spliceosomal components that interact directly with the splice site phosphates have yet to be identified. Neither is it known to what extent the active sites for the two steps of splicing overlap, although growing evidence supports some sort of structural rearrangement between the two steps. Even less is known about the catalytic mechanisms and active site structure of group II introns. Thus, whether the common two-step pathway has resulted from divergent or convergent evolution of these intron types remains debatable. Additionally, what mechanistic similarities exist, if any, between the above introns and introns of the group l self-splicing class are totally unknown. A long-term goal of this laboratory is to elucidate the catalytic mechanisms and active site machinery utilized by the spliceosome and group II introns to catalyze intron excision. This proposal focuses primarily on the exon ligation step. Parallel approaches to be used in both systems include: (l) development of a comprehensive kinetic framework for exon ligation using an assay in which the 3' splice site is added in trans; (2) use of chemically modified 3' splice site RNAs to determine the exact substrate structural requirements for exon ligation, and how these structures contribute to splice site recognition and catalysis; and (3) incorporation of photo- and affinity-crosslinking reagents to identify active site components closely juxtaposed to the phosphodiester backbone at the 3' splice site. Together, these experiments should allow direct comparison of active site structure, 3' splice site recognition and mechanisms of catalysis by the spliceosome and both group II and group I self-splicing introns.